Github repo hosting companion code for "The temporal progression of lung immune remodeling during breast cancer metastasis" (McGinnis et al, 2024, Cancer Cell)

width=200 height=200

Materials include (1) .Robj files for each figure available for download at synapse (www.synapse.org/#!Synapse:syn55388137) and (2) .R files containing custom scripts to reproduce all main text and supplemental figures.

Main Text Figures

Figure 1: Longitudinal scRNA-seq cell atlas of PyMT mouse lung immune cells captures dynamics of the metastatic microenvironment.

• seu_imm_fin_2.Robj - Seurat object of full PyMT longitudinal atlas
• immune_markers.Robj - List of marker genes used to generate cell type annotation dot plot
• jsd_imm.Robj - Jensen-shannon divergence matrix
• anno_freq_summary_imm.Robj - Immune cell type proportion table
• prop_test_imm.Robj - Results of propeller test for significant shifts in immune cell type proportions

Figure 2: AM inflammation and IM wound healing transcriptional signatures are linked to pre-metastatic niche formation.

• seu_int.Robj - Seurat object of IMs in PyMT longitudinal atlas
• seu_alv.Robj - Seurat object of AMs in PyMT longitudinal atlas
• anno_freq_summary_int.Robj - IM subtype proportion table
• anno_freq_summary_alv.Robj - AM subtype proportion table
• crip1_zscores.Robj - Z-score values for Crip1-high Cav1+ signature genes in IM subypes
• umap_alv.Robj - UMAP cell embedding for AMs

Figure 3: NMF and GSEA links Cd14+ inflammatory AM signature to TLR-NFkB inflammation and CD14+ ‘activated’ MDSCs.

• seu_alv.Robj - Seurat object of AMs in PyMT longitudinal atlas (from Fig 2)
• seu_alv_sub.Robj - Seurat object of subsetted 'lipid', 'allergy', and 'antigen' AMs in PyMT longitudinal atlas
• anno_freq_summary_alv_inf.Robj - Inflammatory AM subtype proportion table
• nmf_alv.Robj - Non-negative matrix factorization results for AMs
• gsea_alv_inf.Robj - Gene set enrichment analysis results for CD14+ TLR-NFkB inflammatory AM signature genes (NMF8)

Figure 4: BM-derived myeloid subtype characterization uncovers metastasis-associated DC subtype frequency shifts and TLR-NFkB inflammation in neutrophils.

• seu_mono.Robj - Seurat object of monocytes in PyMT longitudinal atlas
• seu_dc.Robj - Seurat object of DCs in PyMT longitudinal atlas
• seu_neu.Robj - Seurat object of neutrophils in PyMT longitudinal atlas
• seu_neu_mature.Robj - Seurat object of subsetted mature neutrophils in PyMT longitudinal atlas
• anno_freq_summary_mono.Robj - Monocyte subtype proportion table
• anno_freq_summary_dc.Robj - DC subtype proportion table
• anno_freq_summary_neu.Robj - Neutrophil subtype proportion table
• umap_neu_mature.Robj - UMAP cell embedding for mature neutrophils
• mature_neu_zscores.Robj - Z-score values for metastatic stage markers in mature neutrophils

Figure 5: Myeloid TLR-NFkB inflammation signature is detected in PyMT pre-metastatic niche myeloid cells, human metastasis-associated macrophages, and multiple mouse models of metastatic breast cancer.

• seu_mono.Robj - Seurat object of monocytes in PyMT longitudinal atlas (from Fig 4)
• seu_int.Robj - Seurat object of IMs in PyMT longitudinal atlas (from Fig 2)
• seu_mam_sub.Robj - Seurat object of brain MAMs from Gonzalez et al
• nmf_myl.Robj - Non-negative matrix factorization results for myeloid cells
• anno_freq_summary_mono_inf.Robj - Inflammatory monocyte subtype proportion table
• anno_freq_summary_int_inf.Robj - Inflammatory IM subtype proportion table
• gsea_myl_inf.Robj - Gene set enrichment analysis results for CD14+ TLR-NFkB inflammatory myeloid signature genes (NMF19)
• myl_facs_data_summary.Robj - Myeloid CD14 flow data summary for visualizing geometric mean box plots
• myl_facs_data_raw.Robj - Raw myeloid CD14 flow data for visualizing CD14 membrane abundance density plots
• seu_fresh_mono.Robj - Seurat object of monocytes in fresh PyMT validation cohort
• seu_fresh_neu.Robj - Seurat object of neutrophils in fresh PyMT validation cohort
• seu_4t1_mono_fin.Robj - Seurat object of monocytes in 4T1 longitudinal atlas
• seu_4t1_baso_fin.Robj - Seurat object of basophils in 4T1 longitudinal atlas

Figure 6: Lymphocyte subtype characterization reveals details of the inflammatory and immunosuppressive lung metastatic microenvironment.

• seu_nk.Robj - Seurat object of NK cells in PyMT longitudinal atlas
• seu_t.Robj - Seurat object of T cells in PyMT longitudinal atlas
• seu_b.Robj - Seurat object of B cells in PyMT longitudinal atlas
• seu_nk_kim2020.Robj - Seurat object of NK cells from Kim et al
• anno_freq_summary_nk.Robj - NK cell subtype proportion table
• anno_freq_summary_t.Robj - T cell subtype proportion table
• anno_freq_summary_b.Robj - B cell subtype proportion table
• anno_freq_summary_nk_kim.Robj - Kim et al NK cell subtype proportion table
• nk_facs_data_summary.Robj - NK cell CD11b/CD27 data summary for visualizing NK subtyle proportion box plots
• t_cell_zscores.Robj - Z-score values for functional markers in T cells

Figure 7: Intercellular communication modeling reveals metastasis-associated changes in immune cell signaling network.

• seu_imm_fin_2.Robj - Seurat object of full PyMT longitudinal atlas (from Fig 1)
• seu_int.Robj - Seurat object of IMs in PyMT longitudinal atlas (from Fig 2)
• seu_alv.Robj - Seurat object of AMs in PyMT longitudinal atlas (from Fig 2)
• seu_neu.Robj - Seurat object of neutrophils in PyMT longitudinal atlas (from Fig 4)
• cellchat_list.Robj - List of CellChat objects for each metastatic stage in PyMT longitudinal atlas
• neu_ccl6_deg_table.Robj - Table summarizing differential Ccl6 expression tests for neutrophil subtypes at each metastatic stage
• alv_ccl6_deg_table.Robj - Table summarizing differential Ccl6 expression tests for AM subtypes at each metastatic stage
• int_igf1_deg_table.Robj - Table summarizing differential Igf1 expression tests for Mrc1+ IMs at each metastatic stage

Supplemental Figures

Figure S1: Summaries of MULTI-seq classification and results for cryopreserved PyMT, fresh PyMT, and 4T1 temporal cell atlas datasets.

• cellcount_table_pymt_cryo.Robj - Cell counts per sample for longitudinal PyMT atlas
• cellcount_table_pymt_fresh.Robj - Cell counts per sample for fresh PyMT validation cohort
• cellcount_table_4t1.Robj - Cell counts per sample for longitudinal 4T1 atlas

Figure S2: PyMT validation cohort analysis.

• seu_imm_fresh.Robj - Seurat object of full fresh PyMT validation cohort data
• anno_freq_summary_imm_cryo_fresh.Robj - Immune cell type proportion table for PyMT validation cohort data
• anno_freq_fresh_vs_cryo.Robj - Immune cell type proportion table comparing PyMT longitudinal atlas and fresh PyMT validation cohort datasets
• qpcr_summary_tit.Robj - qRT-PCR data for PyMT tumor titration experiment
• qpcr_summary_exp.Robj - qRT-PCR data for PyMT validation cohort data
• jsd_imm_fresh.Robj - Jensen-shannon divergence matrix for fresh PyMT validation cohort data

Figure S3. Dot and feature plots for tissue-resident macrophage subtype annotation markers.

• seu_int.Robj - Seurat object of IMs in PyMT longitudinal atlas (from Fig 2)
• seu_alv.Robj - Seurat object of AMs in PyMT longitudinal atlas (from Fig 2)
• int_markers.Robj - List of marker genes used to generate IM subtype annotation dot plot
• alv_markers.Robj - List of marker genes used to generate AM subtype annotation dot plot

Figure S4. Dot and feature plots for bone marrow-derived myeloid subtype annotation markers.

• seu_mono.Robj - Seurat object of monocytes in PyMT longitudinal atlas (from Fig 4)
• seu_dc.Robj - Seurat object of DCs in PyMT longitudinal atlas (from Fig 4)
• seu_neu.Robj - Seurat object of neutrophils in PyMT longitudinal atlas (from Fig 4)
• mono_markers.Robj - List of marker genes used to generate monocyte subtype annotation dot plot
• dc_markers.Robj - List of marker genes used to generate DC subtype annotation dot plot
• neu_markers.Robj - List of marker genes used to generate neutrophil subtype annotation dot plot

Figure S5. Metastatic progression is associated with pathological activation and degranulation gene expression signature in mature neutrophils.

• seu_fresh_neu_mature.Robj - Seurat object of subsetted mature neutrophils in PyMT validation cohort
• seu_4t1_neu_mature_fin.Robj - Seurat object of subsetted mature neutrophils in 4T1 longitudinal atlas
• mature_neu_4t1_zscores.Robj - Z-score values for metastatic stage markers in mature neutrophils in 4T1 longitudinal atlas
• mature_neu_fresh_zscores.Robj - Z-score values for metastatic stage markers in mature neutrophils in PyMT validation cohort
• umap_neu_mature_fresh.Robj - UMAP cell embedding for mature neutrophils in PyMT validation cohort
• umap_neu_mature_4t1.Robj - UMAP cell embedding for mature neutrophils in 4T1 longitudinal atlas

Figure S6: Cd14+ inflammatory myeloid cells, excluding DCs, express Ccrl2 and are associated with NMF19.

• seu_mono.Robj - Seurat object of monocytes in PyMT longitudinal atlas (from Fig 4)
• seu_dc.Robj - Seurat object of DCs in PyMT longitudinal atlas (from Fig 4)
• seu_neu.Robj - Seurat object of neutrophils in PyMT longitudinal atlas (from Fig 4)
• seu_int.Robj - Seurat object of IMs in PyMT longitudinal atlas (from Fig 2)
• seu_alv.Robj - Seurat object of AMs in PyMT longitudinal atlas (from Fig 2)

Figure S8: Analysis of TLR-NFκB inflammation signature in PyMT validation cohort tissue-resident macrophages.

• seu_fresh_int.Robj - Seurat object of IMs in PyMT validation cohort
• seu_fresh_alv.Robj - Seurat object of AMs in PyMT validation cohort
• anno_freq_summary_alv_inf.Robj - Inflammatory AM subtype proportion table
• anno_freq_summary_int_inf.Robj - Inflammatory IM subtype proportion table

Figure S9: 4T1 longitudinal atlas analysis.

• seu_4t1_imm_fin.Robj - Seurat object of full 4T1 longitudinal atlas
• seu_4t1_int_fin.Robj - Seurat object of IMs in 4T1 longitudinal atlas
• seu_4t1_alv_fin.Robj - Seurat object of AMs in 4T1 longitudinal atlas
• seu_4t1_neu_mature_fin.Robj - Seurat object of subsetted mature neutrophils in 4T1 longitudinal atlas (from Fig S5)
• seu_4t1_baso_fin.Robj - Seurat object of basophils in 4T1 longitudinal atlas (from Fig 5)
• jsd_4t1_sample_all.Robj - Jensen-shannon divergence matrix for full 4T1 longitudinal atlas
• jsd_4t1_sample.Robj - Jensen-shannon divergence matrix for 4T1 longitudinal atlas after removing outgroup samples
• baso_markers.Robj - List of marker genes used to generate basophil subtype annotation dot plot
• umap_baso_4t1.Robj - UMAP cell embedding for basophils
• anno_freq_summary_imm_pymt_4t1.Robj - Immune cell type proportion table for 4T1 longitudinal atlas
• anno_freq_4t1_vs_pymt.Robj - Immune cell type proportion table comparing PyMT and 4T1 longitudinal atlas datasets
• pt_size_4t1.Robj - Table of primary tumor sizes during 4T1 tumor timecourse
• anno_freq_summary_dc_4t1 - DC subtype proportion table for 4T1 longitudinal atlas

Figure S10. Dot and feature plots for lymphocyte subtype annotation markers.

• seu_t_prolif.Robj - Seurat object of subsetted proliferative T cells from PyMT longitudinal atlas
• seu_nk.Robj - Seurat object of NK cells in PyMT longitudinal atlas (from Fig 6)
• seu_t.Robj - Seurat object of T cells in PyMT longitudinal atlas (from Fig 6)
• seu_b.Robj - Seurat object of B cells in PyMT longitudinal atlas (from Fig 6)
• nk_markers.Robj - List of marker genes used to generate NK subtype annotation dot plot
• t_markers.Robj - List of marker genes used to generate T cell subtype annotation dot plot
• b_markers.Robj - List of marker genes used to generate B cell subtype annotation dot plot
• anno_freq_t_all_vs_prolif.Robj - Proliferative T cell subtype proportion table

Figure S11: Flow cytometry analysis of natural killer cell subtypes during metastatic progression.

• nk_facs_gating.Robj - NK cell CD11b/CD27/Thy1/KLRG1 flow cytomtery data used for density plots in NK subtype gates

Figure S12: Intercellular communication modeling analysis in PyMT longitudinal atlas, 4T1 longitudinal atlas, and PyMT validation cohort datasets.

• seu_imm_fin_2.Robj - Seurat object of full PyMT longitudinal atlas (from Fig 1)
• cellchat_list.Robj - List of CellChat objects for each metastatic stage in PyMT longitudinal atlas (from Fig 7)
• seu_neu.Robj - Seurat object of neutrophils in PyMT longitudinal atlas (from Fig 4)
• seu_alv.Robj - Seurat object of AMs in PyMT longitudinal atlas (from Fig 2)
• seu_fresh_alv.Robj - Seurat object of AMs in PyMT validation cohort (from Fig S8)
• seu_fresh_neu.Robj - Seurat object of neutrophils in fresh PyMT validation cohort (from Fig 5)
• seu_4t1_alv_fin.Robj - Seurat object of AMs in 4T1 longitudinal atlas (from Fig S9)
• seu_4t1_neu_fin.Robj - Seurat object of neutrophils in 4T1 longitudinal atlas (from Fig S5)
• seu_4t1_baso_fin.Robj - Seurat object of basophils in 4T1 longitudinal atlas (from Fig S9)
• alv_fresh_ccl6_deg_table.Robj - Table summarizing differential Ccl6 expression tests for AM subtypes at each metastatic stage in PyMT validation cohort
• neu_fresh_ccl6_deg_table.Robj - Table summarizing differential Ccl6 expression tests for neutrophil subtypes at each metastatic stage in PyMT validation cohort
• alv_4t1_ccl6_deg_table.Robj - Table summarizing differential Ccl6 expression tests for AM subtypes at each metastatic stage in 4T1 longitudinal atlas
• neu_4t1_ccl6_deg_table.Robj -Table summarizing differential Ccl6 expression tests for neutrophil subtypes at each metastatic stage in 4T1 longitudinal atlas